Angiogenesis, the formation of new blood vessels from existing ones, is essential to many physiological and pathological processes. Normally, angiogenesis is tightly regulated by pro- and anti-angiogenic factors, but in the case of diseases such as cancer, ocular neovascular diseases, arthritis, and psoriasis, the process can go awry. (Folkman, J., Nat. Med., 1:27-31 (1995).) There are a number of diseases known to be associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, ocular neovascularisation, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid (or hematopoietic) tumors (such as leukemias and lymphomas). Other diseases associated with undesired angiogenesis will be apparent to those skilled in the art.
Although many signal transduction systems have been implicated in the regulation of angiogenesis, one of the best-characterized and most endothelial cell-selective systems involves the Tie-2 receptor tyrosine kinase that is selectively expressed within the vascular endothelium (referred to as “Tie-2” or “Tie-2R” (also referred to as “ORK”), murine Tie-2 is also referred to as “tek”) and its ligands, the angiopoietins (Yancopoulos, G. D., et al., Nature 407:242-48 (2000); Gale, N. W. and Yancopoulos, G. D., Genes Dev. 13:1055-1066 (1999)).
There are four known angiopoietins; angiopoietin-1 (“Ang-1,” alternatively abbreviated as ANGPT1 or Ang1) through angiopoietin-4 (“Ang-4”). These angiopoietins are also referred to as “Tie-2 ligands” (Davis, S., et al., Cell, § 7:1161-1169 (1996); Grosios, K., et al., Cytogenet Cell Genet, § 4:118-120 (1999); Holash, J., et al., Investigative Ophthalmology & Visual Science, 42:1611-1625 (1999); Koblizek, T. I., et al., Current Biology, S:529-532 (1998); Lin, P., et al., Proc Natl Acad Sci USA, 95:8829-8834 (1998); Maisonpierre, P. C., et al., Science, 277:55-60 (1997); Papapetropoulos, A., et al., Lab Invest, 79:213-223 (1999); Sato, T. N., et al., Nature, 375:70-74 (1998); Shyu, K. G., et al., Circulation, 95:2081-12087 (1998); Suri, C, et al., Cell, 37:1171-1180 (1996); Suri, C., et al., Science, 252:468-471 (1998); Valenzuela, D. M., et al., Proc Natl Acad Sci USA, 96:1904-1909 (1999); Witzenbichler, B., et al., J Biol Chem, 273:18514-18521 (1998)).
Both Ang-1 and -2 bind to Tie-2 with an affinity of 3 nM (Kd) (Maisonpierre, P. C., et al., Science 277 (1997) 55-60). Whereas Ang-1 binding to Tie-2 stimulates receptor phosphorylation in cultured endothelial cells, Ang-2 has been observed to both agonize and antagonize Tie-2 receptor phosphorylation (Davis, S., et al., (1996), supra; Maisonpierre, P. C., et al., (1997), supra; Kim, I, J. H. Kim, et al., Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P. C. Maisonpierre, et al., Cardiovascular Research 49(3): 659-70 (2001)). The phenotypes of mouse Tie-2 and Ang-1 knockouts are similar and suggest that Ang-1-stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing vessels in utero through maintenance of endothelial cell-support cell adhesion (Dumont, D. J., et al., Genes & Development, 8:1897-1909 [1994]; Sato, T. N., et al., Nature, 376:10-14 (1995); Suri, C, et al., (1996), supra). The role of Ang-1 in vessel stabilization is thought to be conserved in the adult, where it is expressed widely and constitutively (Hanahan, D., Science, 277:48-50 (1997); Zagzag, D., et al., Experimental Neurology, 59:391-400 (1999)). In contrast, Ang-2 expression is primarily limited to sites of vascular remodeling, where it is thought to block Ang-1 function, thereby inducing a state of vascular plasticity conducive to angiogenesis (Hanahan, D., (1997), supra; Holash, J., et al., Science, 284:1994-1998 (1999); Maisonpierre, P. C, et al., (1997), supra).
Human angiopoietin-2 (“Ang-2,” alternatively abbreviated as ANGPT2 or Ang2) is described in Maisonpierre, P. C., et al., Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998) 389-91. Numerous published studies have purportedly demonstrated vessel-selective Ang-2 expression in disease states associated with deregulated angiogenesis (Bunone, G., et al., American Journal of Pathology, 155:1961-1916 (1999); Etoh, T., et al., Cancer Research, 67:2145-2153 (2001); Hangai, M., et al., Investigative Ophthalmology & Visual Science, 42:1611-1625 (2001); Holash, J., et al., (1999) supra; Kuroda, K., et al., Journal of Investigative Dermatology, 116:113-120 (2001); Otani, A., et al., Investigative Ophthalmology & Visual Science, 40:1912-1920 (1999); Stratmann, A., et al., American Journal of Pathology, 153: 1459-1466 (1998); Tanaka, S., et al., J Clin Invest, 203:34-345 (1999); Yoshida, Y., et al., International Journal of Oncology, 25:1221-1225 (1999); Yuan, K., et al., Journal of Periodontal Research, 35:165-171 (2000); Zagzag, D., et al., (1999) supra). An effective anti-Ang-2 therapy will benefit a vast population of patients with angiogenesis-associated diseases, such as cancer, retinopathies, arthritis, and psoriasis.
A prominent factor also involved in physiological angiogenesis and various diseases and disorders associated with deregulated angiogenesis, for example solid tumor growth, is the vascular endothelial growth factor VEGF-A (also known as VEGF) (Ferrara, Nature (2005) 438, 967-974). Indeed, experiments with neutralizing antibodies and other inhibitors have shown that blockade of the VEGF-A pathway can be sufficient to significantly suppress the angiogenesis associated with tumor growth in many models (Willett, Cancer Cell (2007) 10(2), 145-147; Batchelor, Cancer Cell (2007) 11(1), 83-95) and many therapies targeting this factor have been successful as vascular normalization agents in patients suffering from various conditions arising from pathological angiogenesis, including neovascular age-related macular degeneration (nAMD) (Rosenfeld, N Engl J Med (2006) 355(14), 1419-1431; Trichonas G, Ophtalmol Ther (2013) 2(2), 89-98; Martin, N Engl J Med (2011) 364(2), 1897-1908; Solomon, Cochrane Database Syst Rev (2013) 8, Art.No.:CD005139).
However, recent research on targeted VEGF blockade therapy has revealed that such therapies may promote a more invasive cellular phenotype and enhance tumor cell dissemination (Casanovas, Cancer Cell (2005) 8(4), 299-309; Pae-Ribes, Cancer Cell (2009) 15, 220-231). One theory that accounts for this effect relies on the severe restriction of the oxygen supply to the tumor that occurs when anti-angiogenic agents are used, creating a state of hypoxia. Hypoxia can lead to the transcriptional activation of a number of genes through the stabilization of the HIF-1α transcription factor, which, in the presence of oxygen, is earmarked for proteosomal destruction by oxygen-dependent prolyl-hydroxylases. Targeted genes for activation include VEGF-A, itself, which would, in a normal situation, promote angiogenesis in order to overcome the hypoxia. It has also been reported that Ang-2's expression can be induced by VEGF-A under hypoxic conditions, and may thus further contribute to destabilising vessels in the process of physiological or pathological angiogenesis (Simon, J Cell Physiol (2008) 217(3), 809-818).
More recently, a functional link has been further drawn between Ang-2 and VEGF-A when it was proposed that Ang-2 could be responsible for compensatory tumor revascularization and growth during anti-VEGF therapy and was shown to interfere with anti-VEGFR-2-induced vessel normalization (Bullock, J Clin Oncol (2010) 28, abstr 4630). Data also support a complementary mode of action of antagonists of Ang-2 and VEGF in the context preventing tumor angiogenesis and growth (Hashizume, Cancer Res (2010) 70, 2213-2223).
Given the overlapping and compensatory modes of action of key angiogenic factors with high therapeutic potential such as Ang-2 and VEGF-A, the clinical potential of current monotherapies is clearly limited (Bergers, Nat Rev Cancer (2008) 8, 592-603). Indeed, pre-clinical data recently showed that the simultaneous block of both factors result in enhanced antitumor, antiangiogenic and antimetastatic activity when compared to that of certain monospecific agents used alone (Kienast, Clin Cancer Res (2013) 19(24), 6730-6740). In this context, there is a clear need for the development of potent, dual targeting agents such as the polypeptide disclosed herein.
The present invention satisfies this need and provides anti VEGF-A/Ang2 bispecific therapeutic proteins. Current bispecific antibodies specific for VEGF-A and Ang-2 are, however, sub-optimal, being limited, e.g., by monovalency and geometry, which can impact target engagement and efficacy, not to mention having a relatively high molecular weight of approximately 150 kDa. The present disclosure overcomes these and other limitations by providing novel bi- and multi-specific fusion polypeptides that are at least bivalent for each of the desired therapeutic targets and have unique geometry for improved target engagement.